There has been considerable activity in recent years concerning the design of nucleic acids as diagnostic and therapeutic tools. One aspect of this design relies on the specific attraction of certain oligomer sequences for nucleic acid materials in vivo which mediate disease or tumors. This general approach has often been referred to as “anti-sense” technology. A simplified statement of the general premise is that the administered oligomer is complementary to the DNA or RNA which is associated with, and critical to, the propagation of an infectious organism or a cellular condition such as malignancy. The premise is that the complementarity will permit binding of the oligomer to the target nucleic acid, thus inactivating it from whatever its function might have been, or alter the processing of it to result in alternative product.
Synthetic nucleic acids have been pivotal for the development of life science research, and modified oligonucleotides are developed as a means to treat patients with genetic disease. Most oligonucleotide therapies, including siRNA (short interfering RNA) and antisense technologies (Crooke, S. T. Annu. Rev. Med., 2004, 55, 61-95; Crooke, S. T. Curr. Mol. Med. 2004, 4, 465-487), including splice-switching (Bauman J, et al. Oligonucleotides, 2009, 19, 1-13), are limited by e.g., lability of oligonucleotides in biological fluids and poor delivery to the site of action. Efficiency in regulation of gene expression is more readily achieved if turnover of the target RNA is obtained. This can occur if native enzymes (e.g., RNAse H for antisense and RISC complex for siRNA) can recognise the relevant oligonucleotide complex. A number of diseases cannot be treated by reduction of a specific RNA as some diseases are caused by production of a mis-spliced RNA. For quite a number of such diseases, the RNA produced can be splice-corrected or splice-switched to produce either the correct RNA or an alternative RNA that gives a protein with functions resembling that of the native protein or having other desired properties. Since degradation of the target RNA is not required or even desired there is on the other hand no limitation in modifications due to a need for recognition by cellular degrading enzymes (e.g., RNase H) which opens up the possibility to use oligonucleotides that give tighter binding and high stability to degradation but that are not recognised by such enzymes. A prototype disease for splice-switching therapy is the devastating Duchenne muscular dystrophy (DMD: Moser E. Hum Genet 1984, 66, 17-40; Emery A E. Lancet 2002, 359, 687-695).
A number of oligonucleotide modifications have been explored in the development of oligonucleotides for biotechnology or therapy. 2′-O-alkyloligoribonucleotides (S. M. Freier, K. H. Altmann, Nucleic Acids Res 1997, 25, 4429) is a class of modification that has rendered interest. To modify the 2′-position has several advantages, including low cost starting materials. Compared to 2′-deoxynucleosides, 2′-F- or 2′-O-alkylnucleosides (having electron withdrawing groups in the 2′-position) pushes the conformational equilibrium in the sugar moiety toward the north (C3′-endo) conformations consistent with the A-form geometry of RNA duplexes, which typically leads to more stable duplexes with the target RNA (M. Egli, et al. Biochemistry 2005, 44, 9045). A number of 2′-O-alkyloligoribonucleotide modifications have, as compared to DNA, been shown to give increased stability of duplexes with RNA (E. A. Lesnik, et al. Biochemistry 1993, 32, 7832). Recently the 2′-O-carbamoylmethyl (CM) modification has been studied (M. Grotli, et al. Tetrahedron 1999, 55, 4299) and it was found interesting not least as potential backbone for artificial nucleases and showed that this is highly resistant to enzymatic degradation (S. Milton, et al. Eur. J. Org. Chem., 2012, 539-543).
A single 2′-O—(N-(aminoethyl)carbamoyl)methyl) (AECM) modification in an oligonucleotide resulted in a substantial decrease in melting point of duplexes (H. Ozaki, S. et al. Nucleosides, Nucleotides Nucleic Acids 2009, 28, 943-952). A single AECM modification in a dinucleotide enhanced its stability to nuclease cleavage, and the AECM modification was mentioned as an example of a suitable linker moiety for conjugation to additional substances (U.S. Pat. No. 5,466,786). A favourable nuclease stability of modified oligonucleotides has previously been shown to be in contrast with an efficient cellular uptake of the same (Gary D. Gray, et al. Biochemical Pharmacology, 1997, 53, 1465-1476).